Summary Infection with hepatitis C virus (HCV) is the leading cause of hepatocellular carcinoma (HCC) in patients with liver disease. The mechanisms involved in HCV-related carcinogenesis are not entirely known. Moreover, as new improved treatments for HCV infection emerge, the effect of such treatments in HCV- related HCC is unknown. Our goal is to explore mechanisms of progression to liver cancer by studying hepatocytes of HCV-infected patients through single hepatocyte RNA sequencing during HCV treatment. We hypothesize that infection with HCV will alter gene expression in hepatocytes, and that treatment of HCV will modulate cancer-associated signals. Our preliminary data suggests that human mRNA stabilization by the HCV protein NS5A plays an important role in regulating gene expression, particularly in HCC development. We propose to study the intrahepatic genetic interplay during HCV infection and treatment by performing single hepatocyte genome-wide sequencing in HCV infected patients In specific Aim #1, we will compare mRNA expression in uninfected and HCV-infected single hepatocytes from HCV-infected patients before treatment and uninfected single hepatocytes before treatment to uninfected single hepatocytes after HCV treatment. Through needle aspiration and FACS- directed hepatocyte sorting, we will study whether single hepatocytes infected with HCV have altered mRNA expression compared to uninfected cells from the same patient and its variation following treatment of HCV. In Specific Aim #2, we will determine if NS5A target transcripts involved in cell growth, fibrosis regulation and HCC that we previously identified in HCV-replicon cells exhibit increased expression in HCV-infected hepatocytes, and if increased expression of these cancer-related genes persists, even after HCV treatment. In this aim we will compare and match expression and frequency of cancer-related transcripts in single hepatocytes before and after HCV treatment with those transcript found to specifically bound and stabilize by the HCV protein NS5A during cell culture. The results of the present proposal will provide novel information regarding the influence of HCV on gene expression in the human hepatocyte and will provide the foundation for the identification of possible new therapies and biomarkers for liver cancer following HCV treatment.